JP2016512266A - A new method for protein purification. - Google Patents

Abstract

The present disclosure provides a method for releasing intracellular proteins. The method allows the protein of interest to be separated from the cells without mechanical disruption of the cells, the need for separation of the cells from the culture medium, and the need for removal of the cells from the cell medium.

Description

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH The invention disclosed herein is made in part with government funding and the government has certain rights in the invention. Specifically, some portions of the invention disclosed herein were partially funded under contract number No. N01-AI-60015C by the National Institutes of Health.

The present disclosure relates generally to novel methods and reagents for obtaining intracellular proteins from cell cultures.

Background The concerns surrounding large-scale purification of proteins are an increasingly important issue for the biotechnology industry. Numerous disorders have been the subject of protein or enzyme replacement therapy, including dystrophic epidermolysis bullosa, such as Gaucher disease, Fabry disease and Pompe disease, and lysosomal storage disorders. Large-scale protein production that needs to be supplied to patients must be cost-sensitive, have a production efficiency and produce high quality products. The protein purification process is long and cumbersome and expensive. These disadvantages have a significant impact on the cost of protein replacement therapy and present major challenges for general health.

  Protein production takes place mainly intracellularly, i.e. without mammals, bacteria or fungi engineered to produce the protein by insertion of a recombinant plasmid containing the gene of interest. Cells expressing the protein of interest are cultured in a complex growth medium containing sugars, amino acids and growth factors, usually provided from animal serum preparations. In order to purify a sufficient amount of high quality for use as a human therapeutic, purification requires the separation of the desired protein from the mixture of compounds provided to the cells and from the cell debris.

  Protein purification from cell debris is long and complex. The multiple and repetitive steps necessary to remove the protein of interest can greatly impair the yield and quality of the final protein. In many cases, the protein must be functional after purification.

  Recombinant proteins expressed in the intracellular compartment of a biological expression system are generally released from the cells of the expression system by mechanical disruption in the presence of cell walls. Such mechanical methods include homogenization, microfluidization, nitrogen burst, ultrasound, and bead agitation. Other methods include adding an enzyme to partially break down cell wall components and then adding an osmotic agent to induce rupture and release of periplasmic components. These methods, which combine enzymatic digestion and chemical treatment, are widely used for expressed proteins targeting in the periplasmic space within Gram-negative bacteria. Cells without cell walls will be destroyed by osmotic pressure without the addition of enzymes, or may be completely destroyed by disruption of cell membranes using surfactants or organic solvents. A combination of destructive methods may be used to increase efficiency.

  Most of the previous methods are only suitable for protein release from the periplasmic compartment or completely destroy the cellular compartment. When cells are completely destroyed, DNA can be released from the subcellular compartment and form a viscous liquid. The DNA can be sheared or enzymatically degraded so that the viscosity is reduced and the processing fluid stream can be manipulated during large scale purification. Although these steps have been used successfully for the production of pharmaceutical grade proteins, each processing step adds manufacturing complexity, time and cost.

SUMMARY OF THE INVENTION The present disclosure provides (i) without removing cells from the culture medium, (ii) without using mechanical disruption of the cells, or without using enzymes to disrupt the cell wall; and (iii) Provided are novel methods for purifying intracellular proteins of interest from bacterial cells, particularly E. coli, present in culture media without hardening the cell population into concentrated pellets, and reagents relating to the methods Is.

  The disclosed method comprises a predetermined combination of inorganic salts and solubilizers to solubilize cells for release of recombinant protein without causing total cell disruption that reduces DNA release and increases viscosity. It is used to release intracellular recombinant proteins by adding a surfactant.

  Residual cell debris may be purified away from soluble recombinant protein by selective precipitation after the centrifugation step.

  The method will be accomplished by a series of steps including adding chemical reagents suitable for the bioreactor after completion of the cell culture, ie fermentation. These chemical reagents are added stepwise to the culture. The reagent is also added to achieve a specific concentration of the reagent. The method requires the presence of a chemical reagent in the solution for a specified amount of time.

  The method does not require isolation of cells from the cell culture. Furthermore, it is not necessary to remove the growth medium prior to the addition of the reagent ("free reagent". The method does not use mechanical disruption to lyse the cells.

  In doing so, the method has increased robustness due to lower DNA release while reducing manufacturing complexity, time and cost.

  This document provides a method for releasing a protein of interest from bacterial cells or fungi that express the protein of interest. The method comprises: (a) providing a culture of cells that express the protein of interest (eg, cells that express the protein of interest and a growth medium in which the cells are cultured); (b) the culture of the cells is inorganic. Contacting the salt (eg, by adding a composition comprising an inorganic salt to the culture, etc.); (c) holding the culture containing the added inorganic salt for at least 10 minutes; (d) added Contacting the culture containing the inorganic salt with the chelating agent (eg, by adding a composition containing the chelating agent to the culture, etc.); (e) the culture containing the added inorganic salt and the added chelating agent Holding for at least 10 minutes; (f) adjusting the pH of the culture containing added inorganic salt and added chelating agent to a pH between 4 and 9, depending on the choice. (G) holding the culture containing the added inorganic salt and added chelating agent for at least 15 minutes after pH adjustment; (h) containing the added inorganic salt and added chelating agent Contacting the culture with the surfactant; (i) holding the culture containing the added inorganic salt, the added chelating agent, and the added surfactant for at least 1 hour; (j ) Reducing the temperature of the culture containing the added inorganic salt, added chelating agent, and added surfactant, depending on the choice; (k) added inorganic salt, added chelating agent Contacting the culture containing the added surfactant with the precipitant; (l) added inorganic salt, added chelating agent, added surfactant, and added Holding the culture containing the precipitant for at least 1 hour; and (m) a culture containing the added inorganic salt, the added chelating agent, the added surfactant, and the added precipitant. Performing a method of removing a substantial portion (eg, at least 90%) of cell debris. The method is selected from the group consisting of: (i) mechanical disruption of cells; (ii) removing all or substantially all of the cell medium prior to addition; and (iii) adding an enzyme that digests cell wall material. Does not include at least two steps. In some cases, the method does not include any of (i) mechanical disruption of the cells, (ii) removing substantially all of the cell culture medium, and (iii) addition of enzymes that digest cell wall material. The above method can further comprise removing some or substantially all of the cell culture medium.

  In the above method using an inorganic salt, the inorganic salt may be sodium phosphate, ammonium sulfate, and sodium chloride. In the above method using a surfactant, it may be Triton, SDS, CHAPS 3, Nonidet P40, n-octyl glucoside, and Tween-20. The above method also uses a mixture of two surfactants selected from Triton, SDS, CHAPS 3, Nonidet P40, n-octyl glucoside, and Tween-20. Furthermore, the above method uses PEI and ammonium salts and precipitating agents which may be polyethylene glycol, TCA and ethanol.

  In some cases, the cell that expresses the desired protein is E. coli. The above method uses bacterial cells that are gram negative bacteria. The desired protein is an intracellular protein (ie, it is not secreted from the cell). The desired protein may be DAS181.

  In all the above methods, the step of holding the culture containing the inorganic salt lasts for at least 20 minutes. In all the above methods, the step of holding the culture containing the inorganic salt and the chelating agent may be at least 15 minutes, at least 30 minutes, 45 minutes, or 1 hour, or 30 minutes to 1 hour. .

  In all the above methods, the step of holding the culture containing the inorganic salt, chelating agent and surfactant comprises at least 1 hour, at least 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, It may occur for 6 hours, 7 hours, 8 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours or 5 to 13 hours. In all the above methods, the step of holding the culture containing the inorganic salt, chelating agent, surfactant and precipitating agent comprises at least 1 hour, at least 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, It may occur for 5 hours, 6 hours, 7 hours, 8 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, or 5 to 13 hours.

  The step can be performed when the mixture is at 25 to 35 ° C, preferably 30 ° C. The temperature of the mixture can be lowered to less than 25 ° C., for example between 25 and 20 ° C. to 21 and 23 ° C., prior to addition of the precipitant.

FIG. 1 is a flow chart of an embodiment of a novel protein release protocol. FIG. 2 is a comparison of a protein release method that requires homogenization lysis versus a novel protein release method (“surfactant solubilization”). Figures 3A-3E are SDS PAGE analyzes of the homogenization lysis protocol and the surfactant solubilization protocol. Figures 3A and B show Coomassie detection of proteins isolated from the homogenization protocol (Figure 3A) and the detergent solubilization protocol (Figure 3B). Figures 3C and D show silver detection of proteins isolated from the homogenization protocol (Figure 3C) and the surfactant solubilization protocol (Figure 3D). FIG. 3E is an SDS-PAGE comparison gel with silver detection. In FIG. 3A, lanes 1-10 are: molecular weight marker, clarified homogenate, SP flow-through, SP eluate, HIC eluate, Post HIC TFF, RPC load, RPC eluate, and DAS181 Ref Std 750-, respectively. Contains 0406-002. In FIG. 3B, lanes 1-10 are: molecular weight marker, PEI clarified surfactant, surfactant SP load, surfactant SP flow-through, surfactant SP eluate, surfactant HIC eluate, respectively. Contains surfactant Post-HIC TFF, RPC Load, RPC eluate and DAS181 Ref Std 750-0406-002. In FIG. 3C, lanes 1-10 are: molecular weight marker, clarified homogenate, SP flow-through, SP eluate, HIC eluate, Post HIC TFF, RPC eluate, and DAS181 Ref Std 750-0406-002, respectively. contains. FIG. 4 is a flowchart of a method for optimizing solubilization of a surfactant and a protein purification step. FIGS. 5A-F are analysis of A and B chain proteins. Figures AC are Coomassie staining, Western blot and silver staining analysis of surfactant solubilized yield of Run A, respectively. Lanes 1-10 of these analyzes are as follows: molecular weight marker, SP eluate (Ferm 20110523F2), HIC FT, UF / DF # 1 pool, Capto Adhere FT pH 7.7, titrated Capto Adhere FT pH 5.0, respectively. , Drug substance and DAS181 Ref. Std. FIGS. DF are Coomassie staining, Western blot and silver staining of detergent solubilized yield of Run B, respectively. Lanes 1-10 of these analyzes are as follows: molecular weight marker, SP eluate (Ferm 20110613F2), HIC FT, UF / DF # 1 pool, Capto Adhere FT pH 7.7, titrated Capto Adhere FT pH 5.0, respectively. , Drug substance, and DAS181 Ref. Std. Lot 46-012. 6A and 6B are SDS-PAGE analyzes of DAS181 purity. Shown in FIG. 4A: Lane 1 is 1 molecular weight marker, Lane 2 is drug substance Ferm 20110523F2, Lanes 4 and 5 are drug substance Ferm 20110613F2, and Lane 6 is DAS181 Phase 2 Ref. Std. Lot 46-012. Shown in FIG. 4B are different concentrations of protein: 8-20 μg. 7A-7C show the RP-HPLC comparison of DAS181 from Integrated Run A, Integrated Run B, and DAS Reference Standard. FIGS. 8A-8C show CEX-HPLC comparison of integrated run A, integrated run B and DAS181 from DAS reference. FIG. 9 is an SDS-PAGE analysis of DAS181 stability over several weeks of storage.

DETAILED DESCRIPTION The methods described herein can generally be used to release intracellular proteins from bacterial cells, particularly E. coli. The example described here relates to the release of DAS181 (Malakhov et al., Antimicrob. Agents Chemother., 1470-1479 (2006)) from E. coli. DAS181 is a fusion protein, a heparin (glycosaminoglycan or GAG) binding domain derived from human amphiregulin, and at its N-terminus, Actinomyces viscosus (original language: Actinomyces Viscosus) (for example, SEQ ID NO: 1 ( And fused to the C-terminus of the catalytic domain of SEQ ID NO: 2 (including amino-terminal methionine). The genetically engineered cells described herein contain one or more nucleic acids encoding the DAS181 protein. Suitable cells for in vivo production of DAS181 or recombinant production of any of the polypeptides described herein may be bacterial or fungal.

  Overexpression of the protein in a cell (eg, a bacterial cell) can be achieved using an expression vector. The expression vector may be autologous or integral. Recombinant nucleic acids (such as those encoding DAS181) can be introduced into cells in the form of expression vectors such as plasmids. The recombinant nucleic acid can be maintained extrachromosomally or it can be incorporated into the chromosomal DNA. The expression vector can contain a selectable marker gene that encodes a protein required for cell viability under certain conditions (so that cells transformed with the desired nucleic acid can be detected and / or selected). . The expression vector can also include an autologous replication sequence (ARS).

  Transformed cells (ie, bacterial cells) include, but are not limited to, culturing transformed autotrophic cells in the absence of the necessary biochemical products, selecting and detecting for a novel phenotype Or by culturing in the presence of an antibiotic toxic to yeast in the absence of the resistance gene contained in the transformant. Transformants can be further selected and / or confirmed by integration of the expression cassette into the genome, which can be assessed, for example, by Southern blot or PCR analysis. Prior to introducing the vector into the target cells of interest, the vector can be grown (eg, propagated) in bacterial cells such as E. coli as described above. Vector DNA can be isolated from bacterial cells by any of the methods known in the art in which vector DNA is purified from bacterial milieu. Purified vector DNA can be extracted well with phenol, chloroform, and ether to ensure that E. coli protein is not present in the plasmid DNA preparation. This is because these proteins may be toxic to mammalian cells.

  Expression systems that can be used for small-scale or large-scale production of polypeptides include, but are not limited to, bacteria transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing nucleic acid molecules ( For example, microorganisms such as E. coli), and fungi (eg, S. cerevisiae) transformed with a recombinant fungal expression vector containing a nucleic acid molecule.

  In general, for in vivo production of a protein of interest by a bacterial (eg, E. coli) recombinant cell, the cell is typically in an aqueous nutrient medium containing assimilable nitrogen and carbon sources. Can be cultured under submerged aerobic conditions (shaking culture, submerged culture, etc.). Aqueous media uses a pH of 4.0 to 8.0 (eg 4.5, 5.0, 5.5, 6.0, or 7.5), protein components in the media, buffers incorporated in the media, or acid or base as needed. It can maintain by adding from the outside. Suitable sources of carbon in the nutrient medium include, for example, glucose, sucrose, fructose, glycerol, starch, vegetable oil, petrochemical derived oils, carbohydrates such as succinate, formic acid, lipids and organic acids, etc. Can do. Suitable sources of nitrogen include, for example, yeast extract, corn steep liquor, food extract, peptone, vegetable food, soluble distillate, dry yeast, ammonium sulfate, ammonium phosphate, nitrate, urea, amino acids, etc. Inorganic nitrogen sources can be included.

  Combined advantageous carbon and nitrogen sources need not be used in pure form. This is because low purity substances containing trace amounts of growth factors and significant amounts of mineral nutrients are also suitable for use. Preferred mineral salts such as sodium or potassium phosphate, sodium chloride or potassium, magnesium salt, copper salt can be added to the medium. A small amount of an antifoaming agent such as liquid paraffin or vegetable oil may be added as needed, but is typically not necessary.

Culture of recombinant cells expressing the protein of interest (eg, E. coli) can be performed under conditions that promote optimal biomass and / or enzyme titer yield. Such conditions include, for example, batch, fed batch or continuous culture. Furthermore, changes to the parameters of the conditions can also promote optimal biomass and / or enzyme titer yield of DAS181 protein. Such conditions include, for example, glycerol concentration and high pO ₂ in the culture medium. In order to produce a large amount of biomass, a submerged aerobic culture method can be used, but a small amount can be cultured in a shake flask. For production in large tanks, a number of smaller inoculum tanks can be used to build the inoculum to a level sufficient to reduce lag time in the production vessel. The medium for the production of the biocatalyst is generally sterilized (eg, by autoclaving) prior to cell inoculation. Aeration and agitation of the culture can be accomplished by mechanical means, simultaneous addition of sterile air, or by adding only air in a bubble reactor. For example, larger amounts of pO ₂ (dissolved oxygen) can be used during cultivation in a bioreactor to promote optimal biomass. It can also be used to promote optimal active protein expression in biomass cultures. Implementation of such fermentation parameters, including higher partial oxygen pressure and stepwise glycerol depletion, can increase the production of the protein of interest.

Method of solubilizing surfactant
In-situ surfactant solubilization After sufficient culture for the desired protein expression, the addition of feed into the reactor and the air flow are stopped and the agitation speed is reduced from 100 to 250 rpm, to recover. The temperature is set in the range of 15 ° C to 45 ° C in preparation for the permeabilization step. In some cases, the temperature may be set to 30 ° C. This is also referred to as a pretreatment step in this disclosure. Inorganic salts (eg, sodium phosphate, ammonium sulfate, and sodium chloride) can be added to final concentrations in the range of 10-100 mM. In some embodiments, the final concentration of inorganic salt is 50 mM. Allow the solution to mix for at least 10 minutes or at least 20 minutes. A chelating agent such as ethylenediaminetetraacetic acid (EDTA) is added to a final concentration in the range of 50-200 mM and mixed for at least 10 minutes or at least 20 minutes. In some embodiments, EDTA is added to a final concentration of 100 mM (eg, 50 nM-250 nM). The pH can then be adjusted to 5.0-6.0 (eg, using phosphoric acid, etc.). In some embodiments, the pH is adjusted to 6. Incubate the material for at least 10 minutes (eg, an amount of time ranging from 30 minutes to 180 minutes or more) with mixing (eg, at a mixing speed of 400-450 rpm). In some embodiments of the method, the material is incubated for 60 minutes. A surfactant (eg, Triton, SDS, CHAPS 3, Nonidet P40, n-octyl glucoside, and Tween-20) or a combination of surfactants, eg, sodium dodecyl sulfate (SDS) Triton-X 100, is added to the mixture. Triton-X 100 may be used in combination with 0.01-1% SDS in the range of 2-15%. In some embodiments, 10% SDS solution is then added to a final concentration of 0.1% and Triton X-100 is added simultaneously to a final concentration of 7%. The solution can be mixed at a medium speed at a temperature in the range of 15-45 ^° C. for a time amount ranging from 1 to 5 hours. In some embodiments, the solution is mixed at 30 ° C. at medium speed for 3 hours.

Clarification After incubation in the surfactant combination, the mixture temperature is lowered to 22 ° C. 10 percent (10%) PEI, pH 6.0 stock solution is then added to a final concentration of 0.5%. The solution can be mixed at 22 ° C. for an amount of time ranging from 6 to 24 hours. In some embodiments, the solution is mixed at 22 ° C. for 6-12 hours. In another embodiment of the method, the solution is mixed for 6-8 hours at 22 ° C. The mixture is then clarified by continuous flow centrifugation using Sharples AS-14 (flow rate: 1 L / min). The turbidity (OD600nm) of the mixture is measured at T = 0. Prior to the start of the protein purification process, the mixture can be selectively maintained at ambient temperature overnight.

  Following the surfactant solubilization method described above, the product may be subjected to further purification.

EXAMPLES The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

  The present invention is a novel protein purification protocol that is carried out without requiring mechanical disruption of cells or without enzymatic degradation of the cell wall by an externally added enzyme. It is further contemplated that isolation of the cells from the cell culture is not required and further removal of the cells from the cell culture is not required.

  The new solubilization protocol was optimized to yield a high quality and stable target protein DAS181. This novel protein purification optimization is defined here. The protocol also has the advantage over the usual mode of protein purification, i.e., homogenization, known in the art. The quality, stability and integrity of the protein of interest DAS181 purified through homogenization is compared to that through a novel solubilization protocol. The protein purification protocol utilizes reagents at a specified concentration for a specified time. The optimization of these parameters is discussed below.

Example 1
Protein yield and quality resulting from homogenization as compared to detergent solubilization methods. Extensive research has been conducted to provide the desired protein from the cell as a streamlined alternative to protocols involving mechanical homogenization. The solubilization for in situ release of DAS181 was optimized. Solubilization when compared to homogenization reduces processing time by allowing the release of DAS181 from cells without the steps of cell isolation, cell washing, resuspension, and homogenization. I will. The details of Example 1 describe the solubilization method as compared to homogenization and the subsequent results on protein yield and the quality of the protein after purification. A flow chart showing both the solubilization method and the homogenization method is presented in FIG.

Method Fermentations 20100201 F2 (F2) and 20100201 F3 (F3) were recovered 24 hours after induction. The fermentation was pooled and divided into two aliquots. Each aliquot was solubilized or current homogenized.

Preparation by homogenization method: One aliquot (A) was centrifuged to remove the culture medium. The pelleted cells were suspended in a buffer containing 50 mM potassium phosphate and 200 mM NaCl, pH 8, and then homogenized twice at 15,000 psi on ice. The homogenate was then maintained at ambient temperature until further processing. The homogenate was then treated with polyethyleneimine (PEI; stock 10% PEI, pH 8) to a final concentration of 0.5% and maintained at ambient temperature.

Prepared by solubilization method: The second aliquot (B) was returned directly to the incubator for solubilization at 30 ° C. Dibasic sodium phosphate hepatahydrate and 1M EDTA (free acid at pH 8.7) were added with slow stirring to final concentrations of 50 mM and 100 mM, respectively. The pH was adjusted to 6.0 with 50% HCl and stirring was increased. The mixture was incubated for 1 hour before SDS and Triton X-100 were added to a final concentration of 0.3% and 5%, respectively. Six hours after solubilization of the surfactant, the temperature of the lysate was lowered to 22 ° C., and then 10% PEI, pH 6 was added to a final concentration of 0.5%.

  Proceeding forward, both aliquots were treated equally. Aliquots A and B were incubated overnight with agitation. The homogenate was then titrated with 50% HCl to pH 6 and shaken for approximately 5 hours. Both aliquots were centrifuged and the supernatant was filtered using a further purification method. Clarified surfactant solubilized DAS181 was diluted 1: 1 with H2O for further purification. The homogenate was purified without load dilution of the product for further purification. Samples at each step were measured by OD600nm and DTM CEX-HPLC to determine yield and quality.

  The DAS181 reference material is used as a standard for the following tests. Das181 reference material was purified using a conventional homogeneous protein purification method. Purified DAS181 exists in its active form.

  The turbidity of the centrifuged lysate after PEI treatment was determined before and after further purification. After continuous flow centrifugation, both lysates were found to have similar turbidity at an OD600nm of approximately 0.9. For further purification, the turbidity of the clarified homogenate was observed to be 0.1, whereas the turbidity of the clarified surfactant material was 0.2. The reduced turbidity observed in the homogenate material was attributed to the PEI step performed at pH 8 as opposed to pH 6 in the surfactant step of the solubilization method. In addition, the homogenate material needed to be titrated to pH 6 prior to centrifugation, so the material was less stable over time. The clarified homogenate material maintained at room temperature and 4 ° C. increased in turbidity from 0.1 to 0.2 after 24 hours, while the turbidity of the grown surfactant material remained unchanged. was.

  The yield at each processing step was remarkably similar to both dissolution methods as shown in Table 1. As a result of the surfactant solubilization step, 83% of the total cell protein mass was released, but the yield of the homogenization step was 92%. However, disappearance was observed in the cell centrifugation step prior to homogenization. Therefore, the yield from fermentation to homogenization was 85%. The homogenate PEI precipitation and yield after each column step were similar. The overall homogenization yield was 48%, while the surfactant solubilization yield was 44%.

  Further purification was performed and a summary of product quality is shown in Table 2. The final product of the homogenization swim surfactant solubilization method was shown to be 99.9% and 99.8% monomer, respectively. The homogenate purity was 97.9%, while the detergent pool purity was 98.4%. Further purification showed that the homogenate RT FT pool was 6.5% peak F, 12.6 deamidation (peak C), and 81.0% main peak (peak A). The surfactant pool was 7.3% peak F, 8.2% deamidation (peak C), and 84.5% main peak (peak A). The difference in deamidation is probably the result of homogenization occurring at pH 8 while the surfactant process occurs at pH 6.

Table 2: Comparison of DAS181 quality summary between homogenization and the new “solubilization” method

  Analyzes using further purifications are also performed on the column fractions produced by each lysis method and are summarized in Table 2. The homogenate SP eluate contained approximately twice the amount of HCP as the surfactant SP eluate. The further purified homogenate contained over 40 times the amount of HCP. The homogenates after further purification contained 227 ng / mg HCP and ≦ 16 ng / mg (BLQ), respectively.

  SDS-PAGE analysis showed that the SP eluate generated from homogenization appeared to be slightly less pure than the surfactant SP eluate (FIGS. 3A-3C). Overall, the samples generated by both dissolution methods were similar. The final pool from the homogenization method and the surfactant solubilization method was consistent with the DAS181 reference standard in lanes 9, 10 and 8 of FIGS. 3A, B and C, respectively.

  It was concluded that the overall yield and product quality of the results of the surfactant solubilization and homogenization methods were comparable, except in the lysate produced by the homogenization processing method. Cellular impurities were present. These observations indicate that reduced processing time, improved processing robustness, and possible reduction in host cell impurities are an effective alternative to detergent solubilization methods for homogenization methods for protein purification. It was shown to suggest that there was. These results justified further testing and optimization of the surfactant solubilization method.

Example 2
Optimization of the surfactant solubilization method for protein expression and purification The optimization of the novel surfactant solubilization protocol was to ensure that the purity of the DAS181 drug substance was fully and reproducible. Washing of the SP capture column was made more effective by using a chaotropic agent, guanidine, instead of NaCl to ensure sufficient regeneration. Furthermore, the recovery rate of DAS181 in the SP eluate was improved by increasing the dilution of the centrifugation supernatant before adding Sepharose resin. Hydrophobic interaction chromatography with Hexyl-650C resin was further optimized by increasing the loading capacity. The filtration and polishing steps were replaced with a single chromatographic operation using a multimode strong anion exchange resin.

Method Fermentation samples 20110523F2 and 20110613F2 were collected 24 hours after induction. Agitation was set at 250 rpm. Biphasic sodium heptahydrate and 1M EDTA free acid at pH 8.7 were added to final concentrations of 50 mM and 100 mM. Agitation was increased to 350 rpm and phosphoric acid was added to adjust the pH to 6.0. This mixture was incubated for 1 hour, after which the agitation was increased to 400 rpm and SDS and Triton X-100 were added to final concentrations of 0.1% and 7%, respectively. The temperature of the permeabilized lysate was lowered to 22 ° C. 3 hours after the surfactant permeabilization. Ten percent (10%) PEI of pH 6 was added to a final concentration of 0.5%. This material was incubated for 6 hours and then separated. The resulting supernatant was filtered using a further purification method. The clear surfactant permeabilized lysate was diluted from 125% volume to the final 2 M urea concentration.

Results The final optimized recovery and purification process is summarized in FIG. Two separate operations (Run A and Run B) of the new surfactant protocol were performed in parallel and analyzed to infer optimization of the protocol. Coomassie blue, purified intermediates from Run A (FIGS. 5A-5C) and B (FIGS. 5D-5F), SDS-PAGE by silver staining, and Western blot analysis show that the purity increases as purification proceeds. It has been shown to increase. The final DAS181 drug substance purified from Runs A and B was comparable to the DAS181 reference standard detected by SDS-PAGE by Coomassie Blue. However, with silver staining detection, several slightly stained bands were found below the main bands from the Run A and B samples, indicating impurities. Conversely, Runs A and B had a lower amount of high molecular weight impurities than the reference standard. None of these bands were visible with Coomassie staining detection. Increasing the amount of protein added to the SDS-PAGE / Coomassie Blue analysis (FIG. 6A) revealed that the drug substances from Runs A and B were comparable to the reference standard. One major DAS181 band near 50 kDa was found, but in the close-up image with enhanced contrast, a small band was seen for each added sample just below the major band. This may be an artifact due to excessive addition to the gel. Another gel to which various amounts of reference standards (8 ng to 20 μg) were added demonstrated that SDS PAGE with colloidal blue staining could detect protein additives as low as 20 ng (FIG. 6B). . Lanes with 20 μg drug substance from Runs A and B showed no other bands visible by colloidal blue detection (FIG. 5). Thus, small impurity bands detected using silver staining in the drug substance lane accounted for less than 0.1% of the sample loading (FIGS. 5A-F) because bands reaching 20 ng levels were colloidal. Because it would have been visualized by coomassie blue staining.

  Aggregation analysis after further purification revealed that the drug substances in Runs A and B were 99.8% and 99.9% monomer, respectively (Table 3).

Table 3: Comparative analysis of products from alternative further purification

  Purity analysis by further purification demonstrated that the final drug substance from Runs A and B were 97.1% and 96.7% pure, respectively (Table 4).

Analytical comparison of products from alternative further purification

  The optimized process parameters used in Runs A and B resulted in reduced peak areas and quality samples from further purified impurity peaks (FIGS. 7A-E). In addition, the shoulders away from the main DAS181 peak of drug substance samples from runs # 1 to # 4 were less noticeable than the reference standard.

  The analysis of DAS181 purity and variants obtained with further purification methods, with the exception of deamidation differences, shows a chromatographic profile comparable to the DAS181 reference standard for the final drug substance in the integrated run A and B on a bench scale. (Figure 8). An increase in deamidated DAS181 (peak C) was observed with drug substances from Runs A and B (29.6 ± 2.4% versus 13.2 ± 0.1% of the reference standard). A corresponding decrease in DAS181 (peak A) was also observed. Peak A was determined to be 62.3% and 65.4% from Runs A and B, respectively (Table 5).

Table 5: CEX-HPLC analysis comparison

  These numbers are slightly below the DAS181 reference standard with peak A at 75.5 ± 0.4%. The increase in DAS181 deamidation was attributed to the high pH environment required for certain further purification methods. The percent area of peak C doubled when the pH was maintained from the initial pH of 5.0 to 7.7 for 6-7 hours. The drug substance used in Phase 1 clinical trials was ~ 49% because a number of Phase 1 process steps were performed at pH 8.0 (Table 5, Figure 8F).

  The area of peak 1 representing the misfolded DAS181 was smaller in runs A and B than in the reference standard. The areas of peak 4 and peak F of run A and B drug substance were consistent with the reference standard. Analysis of further purification of the final drug substance produced in Runs A and B was 15 ng / mg and 33 ng / mg as measured (Table 6). These numbers are below or near the limit of quantification (16 ng / mg) for this test. The product level after further purification of the final drug substance was determined to be nearly identical to the level found with the alternative purification product.

Table 6: Comparison of ELISA analysis of products of further purification

  Analysis of sialidase specific activity showed that the final drug substance produced in Runs A and B was 827 U / mg and 847 U / mg, respectively (Table 7). The sialidase specific activity was comparable to the DAS181 reference standard (826 U / mg) run next to the sample for quality control.

Table 7: Comparison of sialidase activity analysis

  Endotoxin analysis of the final drug substance produced in Run A and B integrated on a bench scale measured 0.018 EU / mg and 0.043 EU / mg, respectively (Table 8). The endotoxin specification for the DAS181 drug substance is 0.5 EU / mg. The endotoxin levels of drug substances produced in both runs were far below this maximum. Runs A and B were only 3.5% and 8.7% of the specification limits, respectively.

Table 8: LAL chromogenic endotoxin analysis comparison

  The DAS181 purification recoveries obtained on bench-scale integrated runs A and B were 45.9% and 55.0%, respectively (Table 89. Overall yield is largely related to the outcome of DAS181 release through cell permeabilization and clarification recovery. Affected and a minimum amount of DAS181 material was lost during the chromatographic operation, the predicted step yield did not match the temporary recovery but was higher than expected for the purification operation. Decreased by 3.9 ± 1.5% of the temporary recovery.

Table 9: DAS181 recovery of integrated runs A and B with predicted step yield

  The product recovery after further purification of Runs A and B was 93.1 ± 1.5%. This was an improvement over the product of the alternatively further purified product used in runs # 1 and # 2, which resulted in a recovery of 89.9 ± 2.4%. The yield of UF / DF # 1 work was lower than ~ 88-90% predicted for all bench scale integrated runs. The predicted step yield was exceeded for all chromatographic operations.

  It is concluded that the proposed Phase 3 DAS181 purification process, optimized with improved parameters, resulted in drug substances that were comparable or in some cases better than DAS181 standards in terms of purity, purification recovery, and activity. It was done. The problem of low RP-HPLC purity was previously experienced, but was corrected with Capto Adhere chromatography instead of RP chromatography.

Example 3
Surfactant solubilization protein purification method reagent optimization parameters Various parameters of various detergent solubilization protocols were tested and compared for optimization of the assay based on protein yield. First, various concentrations of Triton combined with a limited range of SDS concentrations were selected to evaluate DAS181 recovery. In addition, various combinations of sodium phosphate and EDTA pretreatments were examined for their effect on DAS181 recovery by detergent solubilization using 7% Triton X-100 and 0.1% SDS at pH 6. In addition, it was determined whether the duration of PEI treatment affected the characteristics of DAS181 during retention times up to 5 days at room temperature. Finally, the stability of the DAS181 clarified surfactant lysate stored at 4 ° C. was evaluated and used to determine acceptable retention times prior to SP chromatography for large scale production.

Method Surfactant concentration analysis: The fermentation broth was aliquoted and subjected to a surfactant solubilization purification method.

  Surfactants were added at various concentrations and stirred at 30 ° C. for 6 hours. The supernatant was subjected to cation exchange HPLC. Relative peak area (% of total peak area) was determined for peaks A, C, and F. The DAS181 concentration was determined by comparing the CEX total peak area to a known concentration standard, and this concentration was normalized to the fermentation recovery determined by the sialidase assay to give the recovery (% of collected) . The selected detergent solubilized sample was clarified by adding 10% PEI (50 mM potassium phosphate, 200 mM NaCl, final pH in 6.0) to reach a final concentration of 0.48% . The supernatant was subjected to turbidity measurement and further purification.

Sodium phosphate and EDTA pretreatment analysis:
The fermentation broth was collected after 24 hours induction. pH 8.7 biphasic sodium heptahydrate and EDTA free acid were added in various concentration combinations. The sample was subjected to a surfactant solubilization protocol. The supernatant was subjected to cation exchange HPLC. Relative peak areas (% of total peak area) were determined for peaks A, C, and F. The DAS181 concentration was determined by comparing the total peak area against a known concentration standard, and this concentration was normalized to the fermentation liquor collection yield to obtain recovery (% of collection).

Evaluation of concentrate that can be permeated after PEI clarification:
The fermentation broth was collected after 24 hours induction. The sample was subjected to a surfactant solubilization protocol. PEI was added to a final concentration of 0.5%. Samples were removed from the fermentor at various intervals. The supernatant was subjected to OD measurement and further purification.
Evaluation of Permeabilization Storage Time after PEI Clarification: The starting material used in this study was a more purified permeabilization product from a fermentation operation. These samples were further purified.

Results Surfactant concentration analysis:
Surfactant Triton and SDS were added in various concentration combinations during the surfactant activated solubilization protocol. The resulting DAS181 yields are summarized in Table 8.

Table 8: DAS181 release after treatment with various concentrations of triton and SDS

Table 8 (continued)

As Triton X-100 concentration was increased from 1% to 8%, there was no clear trend in DAS181 yield due to the change in SDS concentration, so SDS is an important factor in itself,
The concentration factor did not affect the yield. At 8% Triton X-100, there was a clear increase in recovery with a narrower standard deviation with an average yield recovery of 73% across all SDS concentrations. Therefore higher concentrations were investigated. A stable yield was found to be 5-9% Triton X-100. The yield began to decrease at a higher concentration than 9% Triton X-100. A decreasing trend of DAS181 yield at high SDS concentration was also observed. During sampling, surfactant treatments containing more than 9% Triton X-100 were observed to be more viscous than samples with lower Triton X-100 concentrations.

  When samples were treated with PEI, there was a consistent yield at 5-9% Triton X-100 concentration, but the assay varied. A slight decrease in yield was observed with more than 10% Triton X-100. An overall turbidity increase was observed in samples isolated from the protocol with high Triton X-100 concentrations (Table 10).

Table 10: DAS recovery after PEI treatment with various concentrations of surfactant combinations

  The optimal Triton X-100 concentration was concluded to be 5-8%, demonstrating processing latitude to the Triton concentration. The DAS181 yield was consistent with 5-10% Triton X-100, but the turbidity was consistent with 5-8% Triton X-100. These data support the use of Triton X-100 in the 5-8% range combined with 0.05-0.2% SDS.

Sodium phosphate and EDTA pretreatment analysis:
The data show that DAS181 release generally increased with increasing concentrations of sodium heptahydrate and EDTA (Table 11). Furthermore, little or no DAS181 release was observed in control samples that had not been pretreated prior to pH 6 permeabilization. Pretreatment at pH 5 with a given pretreatment concentration of 50 mM sodium phosphate and 100 mM EDTA resulted in DAS181 with approximately the same percent recovery at the same conditions at pH 6 (Table 11).

Effects of various surfactant permeabilizers on DAS181 recovery

  It was concluded that a combination of pretreatment with sodium phosphate and EDTA is necessary to allow pDAS181 E. coli to perform effective Triton X-100 and SDS permeabilization to release DAS181. Overall, these observations suggest that surfactant permeabilization is robust over a wide range of sodium phosphate and EDTA concentrations, pH conditions, and incubation times. The combination of sodium phosphate at pH 6 and 100 mM EDTA was selected as the optimal pretreatment concentration.

Evaluation of PEI clarified permeabilized material:
The turbidity of the supernatant clarified from the PEI treatment, kept at room temperature, increases with increasing retention time (Table 12). The increase in turbidity was greatest in the sample with the shortest PEI treatment time, and the change in turbidity was the smallest in the sample with longer PEI treatment time, so as the PEI treatment time increased, precipitate formation It was confirmed that a clear material with a low content was produced. PEI treatments over 7 hours have the least increase in turbidity within 24 hours, and PEI treatments over 20 hours have a maximum up to the longest measured 120 hours (OD600nm <2.0). Compared with the case where the PEI treatment time exceeded 7 hours, the turbidity OD600nm exceeded 3.0 at 120 hours and the turbidity slightly increased at 24 hours with PEI treatment of less than 6 hours.

  DAS181 recovery was determined immediately after PEI treatment and centrifugation. The recovery ranged from 81 to 95% (Table 4 and Figure 2). Most of the sample points have consistent recoveries between 83-89%. The ratio of DAS181 variants (peaks A, C and F) measured with the fast CEX-HPLC assay was constant within the error of the assay (Table 13). The main DAS181 peak (A) was between 79.93% and 81.63% for all PEI treatments. Peak C was between 10.20% and 12.06%, and peak F was in the range of 7.81% to 8.91%. These data indicate that prolonged PEI treatment does not adversely affect the quality of the DAS181 product.

Table 12: Effect of PEI treatment time and post-clarification retention time on permeabilization turbidity

DAS181 recovery and DAS variant level immediately after PEI treatment

  After a holding time of 24 hours at room temperature, it was concluded that the turbidity of the clarified permeabilizer remained low for all treatment times but was the lowest after 6 hours. After a 48 hour hold, the increase in turbidity is minimal with longer PEI treatment. The ratio of DAS181 variants did not change with PEI treatment time. These data indicate that a PEI treatment time of more than 6 hours allowed for good protein stability for at least 24 hours. This study further shows that the supernatant after PEI clarification can be retained for at least 24 hours without affecting filterability.

Evaluation of storage time for permeabilized PEI growth errors:
The turbidity of the additionally purified post-clarification surfactant permeabilization did not increase after 1 week storage at 4 ° C. (Table 14). The turbidity increased by 46% from the first week to the second week of storage and about 7% from the second week to the third week.

Table 14: Turbidity of surfactant permeabilized product after clarification

  SP FF chromatography recovery was as expected for each run, with some variation in DAS181 disappearance across FT and UTSP washes (Table 15). Almost 5-6% disappearance was observed during further purification, but almost no DAS 181 was observed after further purification.

Table 15: Yield and mass balance for analytical comparison of products after further purification

  For purity analysis by an additional, alternative purification method, SP eluate prepared after 1 to 3 weeks kept at 4 ° C should be similar to SP eluate prepared with fresh feed stock Was shown (Table 16). A relatively high purity was observed over further purification.

Stability of clarified surfactant permeabilizers after further purification summaries.

  Results after further purification showed a slight increase in DAS181 deamidation as seen in the percent increase in peak C in the SP eluate that occurred after 2 weeks of storage (Table 16). This degree of deamidation can be regarded as virtually all T = O samples for surfactant permeabilization storage after clarification, and a recent bench-integrated run sample for further purification. Matches. SDS-PAGE analysis showed consistent stability between the products of alternative further purification (Figure 9). Impurity band intensities looked almost identical to samples after further purification after storage at 4 ° C. for 1, 2, and 3 weeks. The T = O sample appeared to have more impurities than the other samples, which may be due to a slightly more sample being rejected.

  It was determined that there was no reduction in the purity of the sample after further purification that resulted from the feed stock held for up to 3 weeks at 4 ° C. However, CEXHPLC analysis showed a slight increase in deamidation (peak C) after 2 weeks of storage. The clarified surfactant permeabilizer is stable at 4 ° C. for up to 1 week, as shown in more of the further purified samples obtained.

DAS181 array
DAS 181 (without amino terminal Met)
GDHPQATPAPAPDASTELPASMSQAQHLAANTATDNYRIPAITTAPNGDLLISYDERPKDNGNGGSDAPNPNHIVQRRSTDGGKTWSAPTYIHQGTETGKKVGYSDPSYVVDHQTGTIFNFHVKSYDQGWGGSRGGTDPENRGIIQAEVSTSTDNGWTWTHRTITADITKDKPWTARFAASGQGIQIQHGPHAGRLVQQYTIRTAGGAVQAVSVYSDDHGKTWQAGTPIGTGMDENKVVELSDGSLMLNSRASDGSGFRKVAHSTDGGQTWSEPVSDKNLPDSVDNAQIIRAFPNAAPDDPRAKVLLLSHSPNPRPWSRDRGTISMSCDDGASWTTSKVFHEPFVGYTTIAVQSDGSIGLLSEDAHNGADYGGIWYRNFTMNWLGEQCGQKPAKRKKKGGKNGKNRRNRKKKNP (SEQ ID NO: 1)

DAS 181 (with amino terminal Met)
MGDHPQATPAPAPDASTELPASMSQAQHLAANTATDNYRIPAITTAPNGDLLISYDERPKDNGNGGSDAPNPNHIVQRRSTDGGKTWSAPTYIHQGTETGKKVGYSDPSYVVDHQTGTIFNFHVKSYDQGWGGSRGGTDPENRGIIQAEVSTSTDNGWTWTHRTITADITKDKPWTARFAASGQGIQIQHGPHAGRLVQQYTIRTAGGAVQAVSVYSDDHGKTWQAGTPIGTGMDENKVVELSDGSLMLNSRASDGSGFRKVAHSTDGGQTWSEPVSDKNLPDSVDNAQIIRAFPNAAPDDPRAKVLLLSHSPNPRPWSRDRGTISMSCDDGASWTTSKVFHEPFVGYTTIAVQSDGSIGLLSEDAHNGADYGGIWYRNFTMNWLGEQCGQKPAKRKKKGGKNGKNRRNRKKKNP (SEQ ID NO: 2)

Other Embodiments Although the present invention has been described in the context of a detailed description, the foregoing description is for the purpose of illustration and is intended to limit the scope of the present invention. As defined by the scope of the following claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims (18)

A method of releasing intracellular protein of interest from bacterial cells or fungi expressing the protein of interest, comprising:
(A) providing a culture of cells that express the protein of interest within the cell;
(B1) contacting the culture with an inorganic salt; holding the culture containing the added inorganic salt for at least 10 minutes; chelating the culture containing the added inorganic salt Or (b2) contacting the culture with an inorganic salt and a chelating agent;
(C) holding the culture containing the added inorganic salt and added chelating agent for at least 10 minutes;
(D) optionally adjusting the pH of said culture containing said added inorganic salt and added chelating agent to a pH between 4 and 9; and optionally, said added Holding said culture containing inorganic salt and added chelating agent for at least 15 minutes;
(E) contacting the culture containing the added inorganic salt and added chelating agent with a surfactant;
(F) holding said culture containing said added inorganic salt, added chelating agent, and said added surfactant for at least 1 hour;
(G) optionally lowering the temperature of the culture containing the added inorganic salt, the added chelating agent, and the added surfactant;
(H) contacting the culture containing the added inorganic salt, added chelating agent, and the added surfactant with a precipitant;
(I) holding said culture containing said added inorganic salt, added chelating agent, said added surfactant, and said added precipitant for at least 1 hour;
(J) A method of removing a substantial part of cell debris from the culture containing the added inorganic salt, the added chelating agent, the added surfactant, and the added precipitant. Providing a composition comprising the protein of interest, and
At least two steps selected from the group consisting of: (i) mechanical disruption of cells; (ii) removing substantially all of the cell medium; and (iii) adding an enzyme that degrades cell wall material. Not including the way.   Any method selected from the group consisting of (i) mechanical disruption of cells, (ii) removing substantially all of the cell medium, and (iii) addition of enzymes that degrade cell wall material, The method of claim 1, which does not include.   The method of claim 1, wherein the method does not include removing substantially all of the cellular medium.   The method of claim 1, wherein the inorganic salt is selected from sodium phosphate, ammonium sulfate, and sodium chloride.   2. The method of claim 1, wherein the surfactant is selected from the group consisting of Triton, SDS, CHAPS 3, Nonidet P40, n-octyl glucoside, and Tween-20.   6. The method of claim 5, wherein the surfactant is a mixture of two surfactants selected from the group consisting of Triton, SDS, CHAPS 3, Nonidet P40, n-octyl glucoside, and Tween-20.   The method of claim 1, wherein the precipitating agent is selected from the group consisting of PE, and ammonium salts, and polyethylene glycol, TCA, and ethanol.   The method of claim 1, wherein the cell is E. coli.   The method of claim 1, wherein the protein of interest is DAS181.   10. A method according to any preceding claim, wherein step (c) is at least 20 minutes.   11. A method according to any of claims 1 to 10, wherein step (d) is at least 30 minutes, 45 minutes, or 1 hour or 30 minutes to 1 hour.   The step (i) is at least 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours or 2 to 8 hours. the method of.   Step (i) is at least 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 12. A method according to any preceding claim, wherein the time is 12 hours, or 5 to 13 hours.   4. A method according to any of claims 1 to 3, wherein step (g) involves reducing the temperature below 25 ° C.   The method according to any one of claims 1 to 14, wherein step (h) is performed at 20 ° C to 25 ° C, 21 ° C to 22 ° C, or 22 ° C.   The salt of step (b1) or (b2) is at least 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 50 mM, 55 mM, or 60 mM. The method of claim 1, which is a concentration.   2. The method of step 1, wherein the bacterial cell is a gram negative bacterium.   10. The method of claim 1 or claim 9, further comprising the step of purifying the protein of interest from a composition comprising the protein of interest.